•EMB induced chromatin condensation in nuclei and cell apoptosis.•ROS accumulation was increased by EMB whilst lowered by Vitamin E.•DNA damage in Tn5B1-4 cells was mediated by ROS after EMB exposure.•γH2AX-positive cells occurred and caspase-3 expressed in time-dependent manner.Emamectin benzoate (EMB), a novel macrocyclic lactone insecticide, possesses high efficacy and beneficial selective toxicity in agriculture, but so far the EMB-induced cytotoxic action in arthropod insect remains unclear. The present studies were carried out to characterize the property of EMB on the induction of reactive oxygen species (ROS)-mediated DNA damage and apoptosis in Trichoplusia Tn5B1-4 cell model. Following the exposure to EMB at 2.5, 5, 10 or 15 μM, the cells changed to be round, suspended and aggregated, and the decline of cell proliferating ability and cell viability was positively related with the exposure time. Median inhibitory concentration (IC50) of EMB on cell viability was 3.72 μM during 72 h exposure. Apoptosis was induced in 29.8% (24 h) and 39.5% (48 h) of the cells by EMB at 15 μM, showing chromatin condensation in nuclei. The content of ROS in the cells increased rapidly as the concentration of EMB increased, and the pre-incubation of the cells with vitamin E significantly reduced the ROS accumulation. In the treatment of 15 μM EMB, the migrated cell nucleus with DNA strand breaks appeared a teardrop, pear-shaped, or large fan-like tail, and 63.1% of γH2AX-positive cells contained more than four foci, accompanying with high expression level of caspase-3 in time-dependent manner, which consequently led to cell apoptotic death. These evidences in ROS-mediated DNA damage and cell apoptosis induced by EMB may be helpful for deep understanding the cytotoxic action of EMB based on cell model.

•The stem borer possesses tolerance potential to fipronil through changed physiology.•Fipronil at low concentrations provides the selective pressure for larvae tolerance.•The activity of detoxification-related enzymes shows increased response to fipronil.•GAD activity and GABA level in the borers are improved by fipronil exposure.The Asiatic rice borer, Chilo suppressalis Walker, is one of the most destructive rice pests in the world; its physiological adaptability to the sustained application of fipronil was investigated. After topical application, fipronil showed strong lethal effect with LD20 and LD80 values on the fourth instar larvae as 0.00159 and 0.00906 μg per larvae at 48 h- post treatment, and also revealed slight inhibition on the eclosion of pupae and the hatching of eggs. LD20 dosage of fipronil significantly enhanced the activities of carboxylesterase (CarE), glutathione-S-transferase (GST) and cytochrome P450-linked p-nitro O-demethylase (PNOD) in the borers. LD80 dosage of fipronil showed a mild decrease on the activity of CarE, GST and PNOD, and a slight suppression on the activity of glutamate dehydrogenase (GDH), but strongly induced the transform of glutamate to gamma-aminobutyric acid (GABA) and the activity of glutamate decarboxylase (GAD), thereby improved GABA level in the bores. Results suggested that the borer possessed positive tolerance potential to firponil via the modulation of detoxification and GABA responses and provided the basis for further studies on reducing the tolerance of the borer in the control strategies.Download high-res image (156KB)Download full-size image

•Apoptosis of CML K562 and ALL Molt-4 cells occurred under EMB exposure.•Apoptotic cells emerged vitality depression, chromatin condensation and MMP loss.•The excitotoxicity of EMB disturbed intracellular calcium homeostasis.•EMB performanced as an efficacious potential anti-leukemia agent.Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10 μM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48 h exposure to 10 μM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca2+]i level was peaked and persisted within 70 s in K562 cells whilst 50 s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia.Download high-res image (164KB)Download full-size image

•Cytotoxicity of human and insect cells was evaluated under pesticide exposure.•Cytotoxicity varied depending on the pesticides, concentrations and cell type.•Cytotoxicity is a biomarker for pesticide exposure and risk assessment.The cytotoxic potential of 13 commonly used agricultural insecticides was examined using cell-based systems with three human HepG2, Hek293, HeLa cells and three insect Tn5B1-4, Sf-21, and Drosophila S2 cells. Data showed that (1) an enhancement of some insecticides (e.g. pyrethroids) on cells proliferation; (2) an inhibition of some insecticides on cells viability; (3) various levels of susceptibility of different cells to the same insecticide; and (4) the cell type dependent sensitivity to different insecticides. The degree of cytotoxicity of insecticides on human cells was significantly lower than that on insect cells (P<0.05). Methomyl, even 20 μg/ml, showed little cytotoxicity at 24 h exposure whereas emamectin benzoate possessed the strongest cytotoxic potential in a dose-dependent fashion. The results revealed comparable cytotoxic property of agricultural insecticides against intact cells.Download high-res image (206KB)Download full-size image

A series of novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles bearing pyridine ring derivatives was synthesized from 2,6-dimethylpyridin-3,5-dicarboxylic acid diethyl ester. The structures of the compounds were characterized by use of IR, NMR, EI-MS, and elemental analysis. The structure of one compound was further confirmed by X-ray diffraction analysis. Biological assays showed that these compounds had potential insecticidal activity with especially strong regulation of the growth of 2nd instar Mythimna separata larvae. The effects on bioactivity of substituent groups on phenyl ring were also screened.

The hope is that photoactive compounds acting as potential insecticides will have reduced environmental risk, but that is not necessarily the case. In an attempt to elucidate the risk by which photoactivated α-terthienyl (α-T) affects human health, the effects of exposure of human 293 cells and insect Tn-5B1-4 cells to photoactivated α-T at certain doses were characterized. Photoactivated α-T exhibited dose dependence of toxicity and time kinetics of phototoxic activation on the growth of 293 cells (EC50 = 6.23 μg/mL) and Tn-5B1-4 cells (EC50 = 3.36 μg/mL). 293 cells appeared to be anchorage-independent, inflated, and broken; Tn-5B1-4 cells showed significant necrosis. ROS productions and lipid peroxidation of 293 cells were always lower than that of Tn-5B1-4 cells in the treatments of α-T at the same dose. Moreover, photoactivated α-T caused nonselective DNA damage in 293 and Tn-5B1-4 cells at a 10 μg/mL dose and induced cell-cycle progression of 293 cells to increase apoptosis of cells and G1 arrest and decrease in S phase cell population, whereas Tn-5B1-4 cells showed S arrest accompanied by a dose-dependent decrease in G1 and G2 phase cells at a 5 μg/mL dose. These observations suggest that Tn-5B1-4 cells are more susceptible to the action of photoactivated α-T than 293 cells, but photoactivated α-T as an efficient insecticide might be a potential factor in human mutagenic progression.

Twelve novel 2′-hydroxylfurylchalcones have been synthesized by Claisen–Schmidt condensation with galactosylisomaltol, a reagent prepared from lactose. The procedures are environmentally benign and economical. All the compounds are identified by IR, 1H NMR and 13C NMR spectroscopy and by mass spectrometry. Preliminary bioassays indicate that all the title compounds show moderately high herbicidal activities against the height and/or the fresh weight of the seedlings of cucumber, rape, amaranth, wheat, sorghum and Chinese sprangletop at 7.5 g of active ingredient per hm2. However, the compounds exhibit weak fungicidal activities against cucumber powdery mildew, and no activities against rice blast, cucumber grey mould and cucumber downy mildew. The structure–activity relationships are discussed. The present work demonstrates that 2′-hydroxylfurylchalcones could be used as potential lead compounds for further study of novel herbicides.

•Oxa 1 and 2 showed an antifeedant and growth-blocked effect on armyworm larvae.•The more the larvae fed on the Oxa 1 or 2-mixed diets led to the higher mortality.•Midgut α-amylase activity in vivo could be activated or inhibited by Oxa 1 and 2.•Oxa 1 and 2 decreased significantly the midgut α-amylase activity in vitro.1,3,4-Oxadiazoles are a group of diverse pharmaceuticals with a variety of biological activities. The insecticidal activities of 2,5-diphenyl-1,3,4-oxadiazole (Oxa 1) and 1,3,4-oxadiazolyl pyridazinone (Oxa 2) against the armyworm Mythimna separata have been reported. In the present study, we focused on the antifeedant, larval growth regulation, and larvicidal activities of Oxa 1 and 2 against armyworm larvae and the effects of Oxa 1 and 2 on α-amylase in the larval midgut. The structural effects of 1,3,4-oxadiazoles as insecticides were also observed. Longer exposure to increasing concentrations of Oxa 1 and 2 contributed positively to higher antifeedant indexes and slower growth of surviving larvae. In addition, longer feeding times resulted in stronger larvicidal activity. In vivo activation of α-amylase activity in the midgut at 24 h was dependent on the concentrations of Oxa 1 and 2, while longer exposure times contributed to the stronger inhibition of α-amylase activity. Oxa 1 and 2 decreased the in vitro activity of α-amylase in the midgut as significantly as N-bromobutanimide at 5.0 μg/mL. Artificial diets had a more conducive effect on the action of Oxa 1 and 2 than dipped maize leaf. Oxa 1 exhibited a stronger effect on armyworm larvae than Oxa 2. The experiments described here provide information on 1,3,4-oxadiazoles as novel insecticides for use in insect pest control.Download full-size image

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The Km and Vmax experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.

•Sublethal exposure to hexaflumuron changed hemocyte morphology and spreading.•Sublethal exposure altered larval haemogram profile.•Sublethal exposure caused an inhibition on phenoloxidase and nitric oxide synthase.•Sublethal exposure decreased the phagocytic ability of plasmatocytes and granulocytes.Hemocytes circulating in the hemolymph are essential for the insect immunity to protect insects against infections. The effects of sublethal hexaflumuron exposure on the competence of hemocyte immunity of fifth-instar larvae of Mythimna separata were investigated. In this insect, the sublethal exposure could cause plasmatocyte filopodia to contract and shorten, and granulocytes to compact with a loss of cytoplasmic projections in vitro, and induce granulocytes to swell and expand in vivo. The mean number of total hemocytes was significantly declined in feed-thru larvae by 5.0 μg mL− 1 hexaflumuron. Changes in proportional counts of hemocytes showed that sublethal hexaflumuron exposure caused a decrease of granulocytes and an increase of plasmatocytes in a concentration-dependant manner, but these changes were time-dependently reduced. Few effects of the sublethal exposure were revealed on the proportional counts of spherulocytes, oenocytoids, and prohemocytes. The exposure at 24 h showed strong inhibition on phenoloxidase activity in plasma and hemocytes, but this inhibition was time-dependently weakened. The NADPH-diaphorase staining assays showed that a positive immune response of nitric oxide synthase (NOS) in hemocytes was incited by the sublethal exposure, and the longer-time exposure to the higher concentrations of hexaflumuron caused a heavier loss of NOS activity. Phagocytosis rates revealed the inhibitory effect of sublethal hexaflumuron exposure on the phagocytic ability of granulocytes and plasmatocytes that was significantly greater than the effect of chlorpyrifos at the same concentrations. These results show that sublethal hexaflumuron exposure reduces M. separata larval survival by depressing the competence of hemocyte-mediated immune responses.Download high-res image (199KB)Download full-size image

•Emamectin benzoate reduced cell viability and induced a significant increase in cell apoptosis.•Emamectin benzoate caused a formation of single-strand DNA breaks and double-strand DNA breaks.•Emamectin benzoate demonstrated the cytotoxic effect on Sf-9 cells.Emamectin benzoate (EMB), an important macrocyclic lactone insecticide that belongs to the avermectin family and possesses excellent potency in controlling pests, is non-carcinogenic and non-mutagenic conducted in rats and mice, but EMB-induced cytotoxicity and genotoxicity in arthropod insect have been seldom reported yet. In the present paper, we quantified the cytotoxicity of EMB through the detections on cell viability, DNA damage, and cell apoptosis in Spodoptera frugiperda Sf-9 cells in vitro. The results showed that EMB caused a concentration- and time-dependent reduction on the viability of Sf-9 cells, and the median inhibitory concentrations (IC50) were 3.34 μM at 72 h of exposure. The dual acridine orange/ethidium bromide staining showed that exposure to EMB induced a significant time- and concentration-dependent increase on cell apoptosis. The alkaline comet assay revealed that EMB induced significant increases on single-strand DNA breaks, and the percentage of γH2AX-positive cells represented a time- and concentration-dependent formation of DNA double-strand breaks in Sf-9 cells. Interestingly, the similar cytotoxic actions of EMB also went for the human cancerous HeLa cells as a control cell group. Data demonstrated the potential cytotoxic effect of EMB on Sf-9 cells that was significantly greater than the effect of hydrogen peroxide at the same concentrations.Download full-size image

The toxicity and influence on chronic development regulation of dietary benzoxadiazole as well as the subsequent action on cuticle enzyme and antioxidant defense system in feed-thru housefly larvae are investigated. Dietary benzoxadiazole shows limited larvicidal activity and weak interference on larval pupation, but strong blockage against the succedent eclosion process. It does not change the content ratio of protein/chitin in the larval cuticle, but strongly regulates the constituents of cuticle proteins. Moreover, chitinase activities in the integument of third-instar larvae, in vivo, are enhanced and gradually decreased whereas phenoloxidase activities are inhibited and the inhibitory rates are gradually increased. Glutathione S-transferase activities are strongly improved whilst peroxidase activities decrease from about 42.25% to 17.36%, catalase activities decrease from about 80.31% to 27.98% and superoxide dismutase activities are almost unchanged during the different treatment procedures. Peroxidase SDS–PAGE analysis shows that band photodensities of 200.0 and 10.3 kDa proteins in feed-thru larvae are significantly weaker than the corresponding control band. Results suggest that dietary benzoxadiazole might exert strong regulation on larvae cuticle metabolism, interfere with cuticle enzymatic browning and protein sclerotization and weaken the self-protection of larvae against endogenetic oxidative damages.

•Alpha-terthienyl showed light-dependent positive cytotoxicity on Tn5B1-4 and Sf-21 cells.•Photoactivated alpha-terthienyl (PAT) improved significantly ROS level and MDA production.•PAT generated strong oxidative stress to decrease the activity of POD, SOD and CAT.•PAT induced an accumulation of Tn5B1-4 cells in S phase and Sf-21 cells in G2/M phase.Photodynamic sensitizers as useful alternative agents have been used for population control against insect pests, and the response of insect ovarian cells towards the photosensitizers is gaining attention because of the next reproduction. In this paper, antioxidative responses of lepidopteran ovarian Tn5B1-4 and Sf-21 cells to photoactivated alpha-terthienyl (PAT) are investigated. PAT shows positive inhibitory cytotoxicity on the two ovarian cells, and its inhibition on cell viability is enhanced as the concentrations are increased and the irradiation time is extended. Median inhibitory concentrations (IC50) are 3.36 μg/ml to Tn5B1-4 cells, and 3.15 μg/ml to Sf-21 cells at 15 min-UV-A irradiation 2 h-dark incubation. Under 10.0 μg/ml PAT exposure, 15 min-UV-A irradiation excites higher ROS production than 5 min-UV-A irradiation does in the ovarian cells, the maximum ROS content is about 7.1 times in Tn5B1-4 cells and 4.3 times in Sf-21 cells, and the maximum malondialdehyde levels in Tn5B1-4 and Sf-21 cells are about 1.47- and 1.36-fold higher than the control groups, respectively. Oxidative stress generated by PAT strongly decreases the activities of POD, SOD and CAT, and induces an accumulation of Tn5B1-4 cells in S phase and Sf-21 cells in G2/M phase in a concentration-dependent fashion. Apoptosis accumulation of Tn5B1-4 cells and the persistent post-irradiation cytotoxicity are further observed, indicating different antioxidative tolerance and arrest pattern of the two ovarian cells towards the cytotoxicity of PAT.Download high-res image (232KB)Download full-size image